Multiplexed Signal Amplified FISH Via Splinted Ligation Amplification and Sequencing

The present invention relates to a method for amplifying at least one target RNA in a fixed and, optionally, expanded biological sample. In an embodiment of the invention, the method comprises incubating the fixed biological sample with a pair of polynucleotides complementary to non-overlapping and proximal sequences of a target RNA, wherein the polynucleotide pair hybridizes to the target RNA; ligating the polynucleotide pair using a ligase; and amplifying the ligation product. The invention further provides methods for detecting and optionally quantifying and/or sequencing the amplification product. As the method comprises hybridizing polynucleotide pairs to a target RNA in a fixed biological sample, the target RNA can be hybridized in situ.